Carboxylic ionophore (lasalocid A and A23187) mediated lanthanide ion transport across phospholipid vesicles

The transport kinetics of three lanthanide ions (viz., Pr3+, Nd3+, and Eu3+) across dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine unilamellar vesicles mediated by the two carboxylic ionophores lasalocid A and A23187 have been studied by proton nuclear magnetic resonance spectroscopy. Time-dependent changes in the chemical shifts of head group choline signals have been measured to calculate apparent rate constants of transport. These experiments have been done at different ionophore concentrations to determine the stoichiometry of the transporting species. The rates of transport have been found to be faster in the absence of intravesicular La3+ compared to those observed in its presence. The stoichiometry of the transporting species has been found to be 2:1 (ionophore:cation) for both lasalocid A and A23187 in dimyristoylphosphatidylcholine vesicles. However, stoichiometries of greater than 2 have been obtained for lasalocid A mediated lanthanide ion transport across dipalmitoylphosphatidylcholine vesicles. Possible reasons for the observations of such noninteger stoichiometries are discussed. Our results also indicated that A23187 is a more efficient carrier ionophore than lasalocid A.     

Physical characterization of and ionophore-mediated europium(III) transport through unilamellar phosphatidylcholine vesicles. A laser-induced europium(III) luminescence spectroscopy study

A continuation of the study of phospholipid bilayer vesicles as model membrane systems by laser-induced europium(III) luminescence spectroscopy is presented here (B.M. Cader and W. DeW. Horrocks, Jr, Biophys. Chem. 32 (1988) 97). This spectroscopic technique was used to characterize further the physical properties of small and large vesicles composed of dipalmitoylphosphatidylcholine and egg phosphatidylcholine, respectively. Unilamellar preparations were confirmed and internal aqueous volumes were calculated. The calcium-binding carboxylic ionophores, lasalocid A and A23187, were incorporated into the lipid bilayers of these vesicles for the purpose of modeling the mobile carrier mechanism of ion transport across cell membranes. Spectroscopic data implicate the presence of 1:1 and 1:2 europium(III)/lasalocid A complexes within the hydrophobic region, both capable of efficient transport and containing no water molecules in the inner sphere of europium(III). First-order rate constants for lasalocid A-mediated europium(III) transport were determined at 37 and 62 degrees C (0.018 and 0.11 min-1, respectively) using EGTA as a 'flag' to bind and detect the post-transported metal ion.     

23Na-nuclear magnetic resonance investigation of gramicidin-induced ion transport through membranes under equilibrium conditions.

A technique for investigating the gramicidin-facilitated transport of Na+ ions across lipid bilayers of large unilamellar vesicles under the condition of ionic equilibrium has been developed using a combination of heat incubation of the gramicidin with the vesicles and 23Na-nuclear magnetic resonance (NMR) spectroscopy. Isolation of the two 23Na-NMR signals from the intra- and extravesicular Na+ with the shift reagent, dysprosium (III) tripolyphosphate, allows the equilibrium flux of Na+ through the gramicidin channels to be detected and treated as a two-site exchange process. This study indicates that the transport of Na+ through gramicidin channels is second order with respect to the gramicidin concentration.     

Lanthanide-ion transport across phospholipid vesicular membranes: a comparison of alamethicin 30 and A23187 using1H-NMR spectroscopy

The kinetics of Pr³⁺ transport by the ionophores alamethicin 30 and A23187 across unilamellar phospho-lipid vesicular membranes has been compared by following the time-dependent changes in the¹H-NMR spectrum of the vesicles. The measured rates of transport allow stoichiometries of the transporting species to be deduced which are consistent with channel- and carrier-mediated mechanisms respectively. The method provides a useful complement to planar bilayer conductivity studies of these systems.     

Ca2+ ionophores and the activation of human blood platelets: The effects of ionomycin,beauvericin, lysocellin,virginiamycin S,lasalocid-derivatives and McN 4308

Platelet activation is linked to an increase in the cytoplasmic Ca²⁺ concentration and consequently can also be induced by ionophores which mobilize Ca²⁺ from intracellular storage sites or transport it through the plasma membrane. The ionophores mostly used in studies on platelet activation are A 23187 and lasalocid (X-537A). The effects of eight compounds with known Ca²⁺-ionophoric activity in synthetic or natural membrane systems were studied in order to investigate the relationship between transport of Ca²⁺ and activation of platelets.Ionomycin acts as a true Ca²⁺ ionophore: it elicits rapid shape change, aggregation, the release reaction (secretion) and clot retraction (contraction). Beauvericin activates platelets too, but probably not by increasing the cytoplasmic Ca²⁺ concentration. Lysocellin does not activate platelets but induces a passive loss of serotonin. Virginiamycin S has no effect on platelets. Bromolasalocid and one epimer of dihydrolasalocid, like lasalocid, activate platelets by increasing the cytoplasmic Ca²⁺ concentration, and also induce a passive loss of serotonin. McN 4308 does not activate platelets but induces a slow uptake of ⁴⁵Ca²⁺.     

NMR kinetic studies of the ionophore X-537A-mediated transport of manganous ions across phospholipid bilayers

Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn²⁺ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound to two ionized X-537A molecules (Mn X₂). The activation energy for the overall transport process was determined to be 22 ± 5 kcal/mol. Also a pK_a of 5.0 ± 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.     

Ca2+ ionophores inhibit superoxide generation by chemotactic peptide in rabbit neutrophils and the correlation with intracellular calcium

Summary Effects of Ca²⁺ ionophores, A23187 and lasalocid, on superoxide anion generation by chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, in rabbit peritoneal exudate neutrophils were studied. The ionophores by themselves did not activate superoxide anion generation in these neutrophils. When preincubated with the cells for 2 min, both the ionophores inhibited superoxide generation induced by chemotactic peptide. The inhibition was present even in the absence of extracellular Ca²⁺ and the inhibition was better then. Lasalocid produces a dose-dependent chlortetracycline fluorescence decrease response in neutrophils loaded with chlortetracycline. This response is independent of extracellular Ca²⁺ concentration and is related to release of Ca²⁺ from intracellular storage sites. The dose-range at which lasalocid gives this response is same as the dose-range at which it causes inhibition of superoxide response. It may be concluded that the inhibition of superoxide generation by these ionophores is correlated to intracellular Ca²⁺ modulation.Abbreviations FMLP Formyl-Methionyl-Leucyl-Phenylalanine methyl ester     

Interaction of carrier ionophores with phospholipid vesicles

The interactions of carrier ionophores, nonactin, A23187, and lasalocid A with liposomes formed from the synthetic lipids dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine are investigated by differential scanning calorimetry and 1H and 31P nuclear magnetic resonance techniques. The results indicate that the mode of interaction of these ionophores is dependent on the fluidity of the bilayer and on the chemical nature of these ionophores. The 31P NMR studies are suggestive of the formation of small particles that are probably intervesicular lipid-ionophore aggregates in multilamellar vesicles when they are incorporated with these ionophores at high concentrations. The results are interpreted on the basis of the chemical structure and conformations of the ionophores in membrane mimetic media. The 1H NMR line-width measurements indicate that the aromatic rings containing the carboxyl groups of lasalocid A and A23187 are located near the membrane interface while the rest of the molecule is buried in the membrane interior.     

Effect of chain length on the stability of lecithin bilayers

The shift reagent NaCl₃ was added to vesicles of synthetic, saturated (DiC₁₀-C₁₆) lecithins and egg lecithin and the accessibility of the N(CH₃)₃ groups to Na³⁺ ions was studied by NMR. Long chain lecithins, e.g. dipalmitoyl and egg lecithin form bilayers “stable” on the time scale of our experiments and practically impermeable to cations. Short chain lecithins on the other hand form short-lived vesicles surrounded by unstable bilayers which are not effective cation barriers. Ion transport across the latter lecithin bilayers may involve, besides passive diffusion, collision-induced transient rupture and resealing of bilayers coupled with ion movement.     

Energy conservation by Rhodothermus marinus respiratory complex I

A sodium ion efflux, together with a proton influx and an inside-positive ΔΨ, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by ²³Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.     

Characterization of micellar-packaged gramicidin A channels.

The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (¹³C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (²³Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. ¹³C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T₁) demonstrated sodium interaction to be dependent on heat incorporation. The T₁ identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel.     

Ion transport by lasalocid A across red-blood-cell membranes. A multinuclear NMR study

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na-NMR peak areas and chemical shifts, respectively. In similar experiments, Cl- transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge-balance conservation. The effect of disodium 4,4'-diisothiocyanostilbene-2,2'-disulfonate, an anion-exchange inhibitor, has also been studied.     

Studies on growth hormone secretion IX. Prostaglandins do not act like ionophores

To gain further insight on the mechanism of GH secretion in general and on the stimulation of this process by prostaglandins in particular, we compared the effects of PGE₁ and PGE₂ on hormone release and cyclic nucleotide levels with those of the ionophores A23187 and X537A under a variety of experimental conditions. All these substances (in the presence but not in the absence of calcium) enhanced GH release in incubated rat anterior pituitaries , prostaglandins being considerably more potent than ionophores. However, while PGE₂ caused a dose-dependent rise in pituitary cyclic AMP levels (from doubling at 10⁻⁷ M to a two-hundred fold increase at 10⁻⁵ M), the ionophores had no effect on the concentrations of this nucleotide. Neither PGE₂ nor the ionophores had any measurable effect on cyclic GMP levels. Exposure of tissues to ionophores for 60 minutes rendered them refractory to subsequent stimulation by PGE₁ or to ionophores themselves, whereas preincubation with PGE₁ did not diminish GH responses during a second incubation period. On the other hand, 60-minute preincubation of hemipituitaries in the presence of ionophores (10⁻⁵ M) did not suppress subsequent PGE₁-promoted cyclic AMP accumulation. Metabolic blockers inhibited PGE₂ and A23187-promoted GH-release but failed to suppress GH-response to X537A. Verapamil partially inhibited PGE₂ but not ionophore induced GH secretion. Ionophores particularly X537A, accelerated 45Ca efflux while PGE₁ did not influence this. Electronmicroscopy revealed extensive vacuolization localized chiefly at the Golgi apparatus when tissues were incubated with X537A. PGE₁ and A23187 had no such morphological effect. It is concluded that prostaglandins E and ionophores promote GH secretion by different mechanisms.     

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Electrokinetic Phenomena Enhanced Lithium‐Ion Transport in Leaky Film for Stable Lithium Metal Anodes

The application of lithium (Li) metal anodes in Li metal batteries has been hindered by growth of Li dendrites, which lead to short cycling life. Here a Li‐ion‐affinity leaky film as a protection layer is reported to promote a dendrite‐free Li metal anode. The leaky film induces electrokinetic phenomena to enhance Li‐ion transport, leading to a reduced Li‐ion concentration polarization and homogeneous Li‐ion distribution. As a result, the dendrite‐free Li metal anode during Li plating/stripping is demonstrated even at an extremely high deposition capacity (6 mAh cm^?2) and current density (40 mA cm^?2) with improved Coulombic efficiencies. A full cell battery with the leaky‐film protected Li metal as the anode and high‐areal‐capacity LiNi_0.8Co_0.1Mn_0.1O₂ (NCM‐811) (≈4.2 mAh cm^?2) or LiFePO₄ (≈3.8 mAh cm^?2) as the cathode shows improved cycling stability and capacity retention, even at lean electrolyte conditions.     

Comparison of calcium association constants and ionophoretic properties of some prostaglandins and ionophores.

The prostaglandin calcium association constants and calcium transport rates are reported. The calcium association constants for prostaglandins B₂ and E₂ were similar to one another, but lower than those of the ionophores A23187 and X537A. Using a Pressman cell, the ionophores A23187 and X537A, as well as prostaglandin B₂, were found to transport calcium through an organic phase, while the prostaglandin E₂ calcium transport rate was not appreciable in the artifical system.     

Understanding Li‐Ion Dynamics in Lithium Hydroxychloride (Li2OHCl) Solid State Electrolyte via Addressing the Role of Protons

Low‐melting‐point solid‐state electrolytes (SSE) are critically important for low‐cost manufacturing of all‐solid‐state batteries. Lithium hydroxychloride (Li₂OHCl) is a promising material within the SSE domain due to its low melting point (mp < 300 °C), cheap ingredients (Li, H, O, and Cl), and rapid synthesis. Another unique feature of this compound is the presence of Li vacancies and rotating hydroxyl groups which promote Li‐ion diffusion, yet the role of the protons in the ion transport remains poorly understood. To examine lithium and proton dynamics, a set of solid‐state NMR experiments are conducted, such as magic‐angle spinning ⁷Li NMR, static ⁷Li and ¹H NMR, and spin‐lattice T₁(⁷Li)/T₁(¹H) relaxation experiments. It is determined that only Li⁺ contributes to long‐range ion transport, while H⁺ dynamics is constrained to an incomplete isotropic rotation of the OH group. The results uncover detailed mechanistic understanding of the ion transport in Li₂OHCl. It is shown that two distinct phases of ionic motions appear at low and elevated temperatures, and that the rotation of the OH group controls Li⁺ and H⁺ dynamics in both phases. The model based on the NMR experiments is fully consistent with crystallographic information, ionic conductivity measurements, and Born–Oppenheimer molecular dynamic simulations.     

Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems.

Phospholipid vesicles loaded with Quin-2 and 2'',7''-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have been used to investigate the effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin. At an external pH of 7.0, a delta pH (inside basic) of 0.4-0.6 U decreases the rate of Ca2+ transport into the vesicles by severalfold under some conditions. The apparent extent of transport is also decreased. In contrast, raising the pH by 0.4-0.6 U in the absence of a delta pH increases both of these parameters, although by smaller factors. The relatively large effects of a delta pH on the transport properties of Ca2+ ionophores seem to reflect a partial equilibration of the transmembrane ionophore distribution with the H+ concentration gradient across the vesicle membrane. This unequal distribution of ionophore can cause a very slow or incomplete ionophore-dependent equilibration of delta pCa with delta pH. A second factor of less certain origin retards full equilibration of delta pCa when delta pH = 0. These findings call into question several ionophore-based methods that are used to investigate the regulatory activities of Ca2+ and other divalent cations in biological systems. Notable among these are the null-point titration method for determining the concentration of free cations within cells and the use of ionophores plus external cation buffers to calibrate intracellular cation indicators. The present findings also indicate that the transport mode of Ca2+ ionophores is more strictly electroneutral than was thought, based upon previous studies.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Movement of calcium through artificial lipid membranes and the effects of ionophores

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1 : 1 phosphatidylcholine/phosphatidylserine mixture. The percentages of ⁴⁵Ca capture by these three types of liposomes are 10, 1 and 4% respectively.The efflux rates are 2.5 · 10^?6, 2 · 10^?6 and 4 · 10^?5 s^?1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity.The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1 : 2Ca · ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1 : 1 complex, but A23187 as a 2 : 1 complex.